Visualizing Cell State Transition Using Raman Spectroscopy

Citation: Ichimura T, Chiu L-d, Fujita K, Kawata S, Watanabe TM, et al. ( Visualizing Cell State Transition Using Raman Spectroscopy Taro Ichimura 0 Liang-da Chiu 0 Katsumasa Fujita 0 Satoshi Kawata 0 Tomonobu M. Watanabe 0 Toshio Yanagida 0 Hideaki Fujita 0 Laurent Kreplak, Dalhousie University, Canada 0 1 Quantitative Biology Center , Riken, Suita, Osaka , Japan , 2 Department of Applied Physics, Osaka University , Suita, Osaka, Japan, 3 Nanophotonics Laboratory, Riken, Wako, Saitama , Japan , 4 Immunology Frontier Research Center, Osaka University , Suita, Osaka , Japan System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states. - Funding: PRESTO, the Japan Science and Technology Agency (http://www.jst.go.jp/kisoken/presto/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Systems biology is a field of science to understand the biological systems network structure and dynamics rather than just characterizing the function of isolated parts [1]. Advances in computational power and algorithms have pushed systems biology into a new era, enabling to simulate a life of a small organism in silico [2]. Stem cell self renewal and differentiation are attractive targets for systems biology owing to their importance in the life sciences. Systems-level understanding of complex biological system, such as the gene regulatory networks of ESCs, requires comprehensive knowledge of the components and their interactions within a single ESC. However, advances in measurement technology have not yet realized the acquisition of such comprehensive data at the single cell level. As it stands, current systems biology approaches for ESCs thus deals with a limited number of transcription factor networks including the core pluripotency factors [3,4], restricting the understanding of the complicated transcriptional network of ESC. Another approach to understand self renewal and differentiation of ESC is to grasp the changes in the complicated network as whole and visualizing the state transitions on a cell-state landscape. This idea was first introduced by Waddington, where the differentiation potential was drawn as an epigenetic landscape [5], in which the differentiation process is represented as cells rolling down the potential. This type of approach does not necessarily require comprehensive analysis, but often needs quantitative estimations. For example, the cell state can be often estimated by the morphology of the cell, which is also the case in ESC where undifferentiated ESCs form highly packed colonies. Thus, as far as the indices reflect the internal state of the cell, it can be used to describe the state transition of the cell, and accumulated paths of state transition observed in single cell will draw the cellstate landscape. To this end, we focus on Raman scattering microscopy to obtain information of the cell state at the single cell level. The Raman scattering phenomenon arises from molecular vibrations, providing information on chemical species, composition, and the amount of constituent molecules. Thus, Raman scattering imaging can simultaneously detect the location and amount of multiple compounds such as proteins, lipids, DNA, and RNA [6]. Recent advances in Raman scattering microscopy have pushed its applicability to the investigation of biological phenomena in medical and clinical assays for which non-invasive methods are required [7,8]. Since the amount and distribution of the intracellular compounds are related to the cell state, Raman microscopy has been used to describe cell states transitions such as apoptosis, differentiation, and cell division, possibly without harming the cells [9,10,11]. Furthermore, Raman spectroscopy was used to monitor cell state changes after drug exposure [12], during cell cycle [13], and cell differentiation during embryo development [14], showing the capability of Raman spectroscopy in cell state monitoring. In this study, to understand the cell state transition during the differentiation, we performed Raman spectral imaging of mouse ESCs during differentiation, and compared the results with those from terminally differentiated cell-lines including fibroblasts, epithelial cells, and hepatocytes. In addition, cells with differentiation capability, such as bone marrow mesenchymal stem cells (MSCs), adipocytes, and neuroblasts, were analyzed which showed significant changes in the Raman spectra during the differentiation process. By carefully analyzing the Raman spectra of the cellular nucleus, we were able to illustrate the differentiation pathway of the ESCs. In this study, we did not concentrate on deducing the molecular species from Raman spectra but we rather tried to interpret the spectral shapes in a morphological way. Here we propose Raman spectrum morphology, a method for visually understanding the cell state without recurring to labeling strategies, and demonstrate the discrimination of the cell state transition on the landscape by Raman spectra. Materials and Methods Cell culture Mouse ESCs (E14Tg2a) were purchased from the Riken Cell Bank (Ibaraki, Japan) and maintained on feeder-free gelatincoated plates in Leukemia Inhibitory Factor (LIF)-supplemented medium: Dulbeccos modified Eagles medium-High Glucose (DMEM-HG; Invitrogen, Ca (...truncated)